A User-Friendly Approach for Routine Histopathological and Morphometric Analysis of Skeletal Muscle Using CellProfiler Software

Adult skeletal muscle is capable of active and efficient differentiation in the event of injury in both physiological and pathological conditions, such as in Duchenne muscular dystrophy (DMD). DMD is characterized by different features, such as continuous cycles of degeneration/regeneration, fiber heterogeneity, chronic inflammation and fibrosis. A well-defined and standardized approach for histological and morphometric analysis of muscle samples is necessary in order to measure and quantify specific regenerative parameters in myopathies. Indeed, non-automatic methods are time-consuming and prone to error. Here, we describe a simple automatized computational approach to quantify muscle parameters with specific pipelines to be run by CellProfiler software in an open-source and well-defined fashion. Our pipelines consist of running image-processing modules in CellProfiler with the aim of quantifying different histopathological muscle hallmarks in mdx mice compared to their wild-type littermates. Specifically, we quantified the minimum Feret diameter, centrally nucleated fibers and the number of macrophages, starting from multiple images. Finally, for extracellular matrix quantification, we used Sirius red staining. Collectively, we developed reliable and easy-to-use pipelines that automatically measure parameters of muscle histology, useful for research in myobiology. These findings should simplify and shorten the time needed for the quantification of muscle histological properties, avoiding challenging manual procedures.